Project 3: Pathways and partners of p66Shc: functional genomics P66Shc deficiency leads to lifespan extension in mice through an unknown mechanism. Biochemical studies by the Pelicci and Rizzuto groups have demonstrated a stress-dependent localization of p66Shc to mitochondria, and have demonstrated effects of p66Shc on ROS generation, mitochondrial mutagenesis, apoptosis, angiogenesis, adipogenesis and body size. At every point that it has been tested, the redox function of p66Shc has been essential for biochemical or biological activity. Thus, the major hypothesis for the means by which p66Shc deficiency extends lifespan is that it is through its biochemical redox function, which appears to have consequences on ROS generation, apoptosis, and angiogenic and adipogenic functions. Microarray technology has over the past few years become an important method for verifying existing hypotheses and generating new hypotheses. The precision and reliability of the microarray reagents and hardware has increased, and high- level analytical software packages have been developed to identify transcriptional emphasis on particular metabolic pathways. Thus we propose an intensive microarray analysis; the preliminary data already suggests mechanisms by which p66Shc deficiency inhibits ROS generation, adipogenesis, and angiogenesis (see Preliminary Results). We have recently used a similar microarray analysis to identify the likely function of the mitochondrial protein frataxin in human cells (Tan et al., HMG 2003) and some major transcriptomal and metabolic consequences of its depletion, which were confirmed by biochemical and proteomic tests (Schoenfeld et al., HMG 2005). So, by analogy, we propose to use microarray and proteomics to identify the major function of p66Shc gene and the mechanisms by which p66Shc deficiency extends lifespan. Thus, we will perform Affymetrix-based microarray analysis of the genomic effects of p66Shc deficiency in liver, muscle, heart, lung, and adipose tissue as well as in mouse embryo fibroblasts of mutant and control cells, with and without oxidative stress. We will compare these data to our large microarray database of transcriptomal changes that occur as a result of mitochondrial perturbation by mutations or biochemical inhibitors. We will also perform a proteomic analysis of p66Shc (-/-) cells using two kinds of 2-dimensional gel electrophoresis to confirm the microarray analysis. Relevance to public health: The conserved nature of the IGF-lnsR axis pathway for lifespan extension from flies to worms to mice, and the special focus of p66Shc on adiposity, suggests that elucidation of the p66Shc will likely be relevant to human health in the United States.